The pathogenesis and biochemistry of amyloidosis

The transformation of serum proteins into Congo red-sensitive fibrillar material is requisite for the onset and progression of amyloid disease. All the mechanisms which lead to the disease itself have not been elucidated, but our knowledge has increased significantly. It is apparent that in all types of amyloid fibrils, three common features are displayed by the major protein constituents. These are that the fibril protein has a serum precursor, a high degree of anti-parallel beta-sheet conformation and a distinctive ultrastructure on electron microscopy. In the AL and AA forms of amyloidosis, the putative precursors appear to undergo limited degradation to form the protein component of amyloid fibrils. It has been suggested that there may be certain primary structural characteristics inherent in precursor molecules which make them amyloidogenic, thus predisposing them to amyloid fibril formation. This would include certain subtypes of immunoglobulin light chains, possibly kappa I and lambda VI, in the AL type of amyloidosis and one of the polymorphic SAA species, SAA2, which has been identified as the predominating isotype found in AA amyloid fibrils. In AH amyloidosis, the mechanism of amyloid fibril formation appears to be simply a concentration phenomenon where elevated concentrations of B2-M are not handled normally and amyloid deposition is the result. Amyloidogenesis in the hereditary form of systemic amyloidosis may involve other factors in addition to the presence of a variant precursor prealbumin as indicated by the delayed onset of the disease. It is evident that the elucidation of the mechanism(s) which governs the onset and progression of the amyloidoses will allow future regulation and treatment of these all too often complex disorders.     

Diagnosis and treatment in systemic amyloidosis

Systemic amyloidosis is characterized by the involvement of multiple organs and the presence of an amyloid precursor protein in serum. This disorder is classified into four major forms: immunoglobulin light chain-derived (AL), reactive AA, dialysis-related (beta2M) and hereditary transthyretin (ATTR) type. Heart, kidney, gastrointestinal tract and peripheral nerves are commonly affected by amyloid deposition in systemic amyloidosis and histopathological demonstration of amyloid deposits on any of affected organs is the first step leading to the diagnosis of this disease. Immunohistochemical analysis of amyloid protein on tissue amyloid deposits is necessary to make classification of the disease and DNA testing is also useful in a hereditary form. Amyloidosis had been considered to be an incurable disease but during the past one decade several therapeutic approaches have been employed for the amyloidosis patients with diverse pathogenetic backgrounds: intravenous large dose of melphalan accompanied by autologous peripheral blood stem cell transplantation for AL amyloidosis and liver transplantation for hereditary ATTR type amyloidosis. As a result some amyloidosis patients have been rescued and are now enjoying their own social lives. It is likely that recent advance on the research of amyloidosis has changed the concept of this disease.     

Amyloidosis

Amyloidosis is a heterogeneous group of disorders characterized by extracellular deposition of abnormal protein fibrils which are dérived from different proteins in different forms of the disease. Asymptomatic amyloid deposition in a variety of tissues is a universal accompaniment of ageing, and clinical amyloidosis is not rare. Intracerebral and cerebrovascular β-protein amyloid deposits are a hallmark of the pathology of both sporadic and familial Alzheimer's disease, β₂-microglobulin-derived amyloid is a common complication of long term haemodialysis, and islet amyloid polypeptide is the fibril protein in the universal islet amyloidosis of type II diabetes mellitus. New fibril proteins have lately been identified in hereditary amyloidosis, including variants of gelsolin, apolipoprotein AI, lysozyme and fibrinogen. The development of radiolabelled serum amyloid P component (SAP) scintigraphy has allowed amyloid to be diagnosed non-invasively in vivo for the first time, provided unique insight into the distribution and size of amyloid deposits, and yielded novel information on the natural history and the effects of treatment. Amyloid deposits are in a state of dynamic turnover and can regress if new fibril formation is halted. The recent elucidation of the three dimensional structure of human SAP may enable the design of specific therapeutic agents.     

Livers from patients with apolipoprotein A-I amyloidosis are not suitable as "domino" donors.

Orthotopic liver transplantation, by eliminating the major site of amyloidogenic protein synthesis, is currently the only definitive treatment of most hereditary amyloidoses. Because of the minimal parenchymal involvement, the explanted livers from familial amyloidotic polyneuropathy (FAP) patients have been transplanted into non-FAP patients in a "domino" fashion. The aim of this study was to evaluate the extent of amyloid deposits in explanted livers from two patients with apolipoprotein A-I amyloidosis, with the Arg26 mutation, to determine their suitability as domino donors. A detailed histologic review of the explanted livers from two patients was performed and assessed for the extent of amyloid deposition by routine and Congo red stains. Both patients had identical histopathologic features. The liver parenchymal involvement was strikingly severe. Large patches of amyloid separated hepatic cords, with accentuation around the central veins. All portal triads were consistently and markedly involved with amorphous eosinophilic deposits within the connective tissue compressing the bile ducts and vascular structures. Hilar vessels had patchy deposits. No involvement of hilar nerve branches was seen. The hepatic parenchyma is extensively involved in hereditary Apolipoprotein A-I amyloidosis, with the Arg26 mutation. These livers, removed at orthotopic liver transplantation, are not suitable for domino donation.     

Diagnosis and immunohistochemical classification of systemic amyloidoses

 Fourty-three cases of systemic amyloidosis were identified in an unselected autopsy series from our institute (6305 autopsies between 1979 and 1993) and classified immunohistochemically by means of a panel of antisera directed against five major amyloid fibril proteins. Amyloid A (AA) amyloidosis was the most common type, being found in 21 cases (48.8%). Transthyretin-derived (ATTR) amyloidosis was present in 11 cases (25.6%), and immunoglobulin light chain-derived (AL) amyloidosis in 10 cases (23.3%). A single case (2.3%) contained deposits of more than one type of systemic amyloid. AA amyoloidosis was associated with chronic inflammatory or infectious diseases (81%), malignant tumours (19%) or both (9.5%). Immunoglobulin light chain-derived amyloidoses were associated with myeloma (50%) or primary (idiopathic; 50%). In AA and AL amyloidosis the kidney was the organ most frequently involved. ATTR amyloid affecting mostly the heart and lungs presented as senile systemic amyloidosis. Systemic amyloidosis was the cause of death in 5 cases (12%) and caused symptoms in 17 cases (39%). Our results suggest that most cases can be classified by using a panel of sensitive and specific antibodies against five major amyloid fibril proteins. This technique may make amyloid type-specific therapy possible for AL amyloid patients who do not have evidence of an underlying plasma cell dyscrasia. Received: 9 December 1997 / Accepted: 2 February 1998     

Microdeposits of amyloid in sclerocalcific heart valves: a histochemical and immunofluorescence study.

Amyloid associated with seven sclerotic and two normal aortic and mitral valves was studied. The sclerotic valve amyloid contained microfibrils with typical random orientation and a fibril width of 9.5-12.5 nm. The amyloid deposits demonstrated permanganate-resistant Congophilia and contained the amino acid tryptophan. Immunofluorescence studies showed P-component in amyloid deposits of 6 of 7 valves, but none of the sclerotic valves contained amyloid fibril proteins of the AL (primary), AA (secondary), AEt (medullary thyroid carcinoma) or ASc1 (senile cardiac) types. Two non-sclerotic valves, removed from a patient with systemic amyloidosis, showed permanganate-sensitive Congophilic amyloid deposits which contained amyloid fibril protein AA.     

Bone marrow findings correlate with clinical outcome in systemic AL amyloidosis patients

AIMS: Bone marrow sampling is a key investigation in the work-up of amyloid light chain (AL) amyloidosis, but the relationship between bone marrow findings and the varied phenotype and clinical outcome of AL amyloidosis is unclear. The aim was to determine if bone marrow pathological parameters at diagnosis were related to clinical behaviour in AL amyloidosis patients. METHODS AND RESULTS: Bone marrow findings, clinical features and outcome of 80 patients referred with a diagnosis of systemic AL amyloidosis were evaluated; six patients were subsequently excluded due to re-categorization as other forms of amyloidosis. At latest follow-up (median 66 months), 11 of the 18 patients with no identifiable bone marrow neoplastic cells (61%) versus only seven of the 56 patients with neoplastic plasma cells or non-Hodgkin's lymphoma (13%) were alive (P = 0.0046). However, neither the quantity of the neoplastic cells nor the serum light chain levels were correlated with amyloid burden or patient survival. CONCLUSIONS: Identification of a neoplastic population in the bone marrow of AL amyloidosis patients by histology and immunohistochemistry correlates with poor outcome; however, the neoplastic cell burden is not prognostically significant, suggesting that additional factors are important in determining disease behaviour in AL amyloidosis.     

Amyloid protein of vessels in leptomeninges, cortices, choroid plexuses, and pituitary glands from patients with systemic amyloidosis

The cerebrum, cerebellum, and choroid plexuses from 16 patients with systemic amyloidosis, and the pituitary glands from 14 of these patients, were investigated histologically and immunohistochemically. Cerebrovascular amyloid (CVA) was found in the leptomeninges and cortices of six patients with systemic amyloidosis, including two patients with amyloid A protein (AA) amyloidosis related to serum amyloid A protein, one with AL amyloidosis related to immunoglobulin light chain (AL), two with familial type I amyloidotic polyneuropathy (FAP), and one with senile systemic amyloidosis (SSA). CVA protein from two patients with FAP reacted with anti-human prealbumin antibody similar to that of the visceral organs of these two patients. CVA in SSA reacted with anti-human prealbumin antibody and anti-beta protein antibody. Vascular amyloid was frequently noted in the pituitary glands and choroid plexuses of patients with systemic amyloidosis, and was found to be identical to that in the visceral organs (heart, kidney, and intestine) of these patients. CVA in the leptomeninges and cortices from two patients with AA amyloidosis and one with AL amyloidosis reacted with anti-beta protein monoclonal antibody but not with anti-human AA monoclonal antibody, anti-human A lambda antisera, and anti-human A kappa antisera. We suggest that amyloid proteins of AA and AL amyloidosis do not readily accumulate in the vessels in the leptomeninges and cortices even though the proteins circulate, and that beta protein is not derived from a serum precursor.     

Amino Acid Sequences in Amyloid Proteins of χIII Immunoglobulin Light-Chain Origin

The main amyloid fibril (AL) proteins extracted from the spleen of Patient So 124 with systemic amyloidosis and from a skin nodule of Patient KSA with localized amyloidosis were studied by partial amino acid sequence analysis and proved to be of kappa III immunoglobulin light-chain origin. The sequences were similar to that of Bence Jones protein V and, which has been reported to have a unique kappa III subset sequence. Thus, except for position 9 in protein AL(KSA), the amino acid sequences were identical to position 25 in AL(So 124) and in AL(KSA). The question is being raised whether this kappa III subset might contain amyloidogenic sequences.     

Bronchioloalveolar carcinoma and generalized amyloidosis complicating progressive systemic sclerosis.

We report a patient with progressive systemic sclerosis in whom an autopsy, performed 13 years after diagnosis, revealed the presence of bronchioloalveolar carcinoma and of generalized amyloidosis. Characterization of the amyloid fibril protein suggested an immunoglobulin light chain (AL) origin.     

A putative role for cathepsin K in degradation of AA and AL amyloidosis

The aims of this study were to investigate the role of cathepsin K in the pathology of amyloidosis by demonstrating its presence in multinucleated giant cells (MGCs) adjacent to amyloid deposits, and determining its ability to degrade amyloid fibril proteins in vitro. The study was performed using autopsy and biopsy specimens from patients with AA or AL amyloidosis. In six (55%) patients with AA amyloidosis and seven (58%) patients with AL amyloidosis, variable numbers of CD68-immunoreactive MGCs were found adjacent to amyloid deposits. In each case strong cytoplasmic immunostaining for cathepsin K was found in MGCs; immunostaining of amyloid deposits was present in five (45%) patients with AA amyloidosis and three (25%) patients with AL amyloidosis. In vitro degradation experiments showed that recombinant cathepsin K completely degraded AA amyloid fibril proteins at pH 5.5 as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Less effective degradation took place at pH 7.4 and there was no degradation in the presence of a general cysteine protease inhibitor (E64) or in the absence of cathepsin K. This is the first study to show that cathepsin K is expressed in MGCs adjacent to amyloid deposits and to demonstrate its ability to degrade amyloid fibril proteins.     

Disease-associated prion protein is not detectable in human systemic amyloid deposits

Cerebral and cardiac amyloid deposits have been reported after scrapie infection in transgenic mice expressing variant prion protein (PrP(C)) lacking the glycophosphatidylinositol anchor. The amyloid fibril protein in the systemic amyloid deposits was not characterized, and there is no clinical or pathological association between prion diseases and systemic amyloidosis in humans. Nevertheless, in view of the potential clinical significance of these murine observations, we tested both human amyloidotic tissues and isolated amyloid fibrils for the presence of PrP(Sc), the prion protein conformation associated with transmissible spongiform encephalopathy (TSE). We also sequenced the complete prion protein gene, PRNP, in amyloidosis patients. No specific immunohistochemical staining for PrP(Sc) was obtained in the amyloidotic cardiac and other visceral tissues of patients with different types of systemic amyloidosis. No protease-resistant prion protein, PrP(res), was detectable by Western blotting of amyloid fibrils isolated from cardiac and other systemic amyloid deposits. Only the complete normal wild-type PRNP gene sequence was identified, including the usual distribution of codon 129 polymorphisms. These reassuringly negative results do not support the idea that there is any relationship of prions or TSE with human systemic amyloidosis, including cardiac amyloid deposition.     

Morphologic and chemical variation of the kidney lesions in amyloidosis secondary to rheumatoid arthritis

The kidneys of 20 patients who died of secondary systemic amyloidosis due to rheumatoid arthritis were studied histologically, and four of these were shown to have an uncommon pattern of deposition with almost no glomerular involvement but heavy deposits in the outer zone of the medulla. In three of the four patients frozen tissue was available. Immunochemical characterization of amyloid fibrils from these three cases showed that the major subunit amyloid fibril protein was protein AA, typical of secondary amyloidosis. Gel chromatography of fibrils revealed an uncommon elution pattern with two retarded major protein peaks. Both these proteins showed immunologic identity with protein AA and had N-terminal amino acid sequences identical with that protein but differed in size obviously due to a shortening of the C-terminal in one of the proteins. The reason for the correlation between the pattern of deposition of amyloid and alterations in protein AA is unclear but might be due to variations in enzymes responsible for the cleavage of the amyloid fibril subunit precursor protein SAA.     

Evaluation of systemic amyloidosis by scintigraphy with 123I-labeled serum amyloid P component

BACKGROUND. In systemic amyloidosis the distribution and progression of disease have been difficult to monitor, because they can be demonstrated only by biopsy. Serum amyloid P component (SAP) is a normal circulating plasma protein that is deposited on amyloid fibrils because of its specific binding affinity for them. We investigated whether labeled SAP could be used to locate amyloid deposits. METHODS. Purified human SAP labeled with iodine-123 was given intravenously to 50 patients with biopsy-proved systemic amyloidosis--25 with the AL (primary) type and 25 with the AA (secondary) type--and to 26 control patients with disease and 10 healthy subjects. Whole-body images and regional views were obtained after 24 hours and read in a blinded fashion. RESULTS. In the patients with amyloidosis the 123I-SAP was localized rapidly and specifically in amyloid deposits. The scintigraphic images obtained were characteristic and appeared to identify the extent of amyloid deposition in all 50 patients. There was no uptake of the 123I-SAP by the control patients and the healthy subjects. In all patients with AA amyloidosis the spleen was affected, whereas the scans showed uptake in the heart, skin, carpal region, and bone marrow only in patients with the AL type. Positive images were seen in six patients in whom biopsies had been negative or unsuccessful; in all six, amyloid was subsequently found on biopsy or at autopsy. Progressive amyloid deposition was observed in 9 of 11 patients studied serially. CONCLUSIONS. Scintigraphy after the injection of 123I-SAP can be used for diagnosing, locating, and monitoring the extent of systemic amyloidosis.     

ALys amyloidosis caused by compound heterozygosity in exon 2 (Thr70Asn) and exon 4 (Trp112Arg) of the lysozyme gene

Hereditary amyloidoses are caused by germline mutations, which increase the propensity of a protein to form cross-beta aggregates and deposit as amyloid. Hereditary amyloidoses are particularly interesting as they help to understand how changes in the primary structure of an otherwise non-amyloidogenic protein contribute to amyloidogenesis. Here we report on a novel form of systemic ALys amyloidosis, caused by compound heterozygosity in exon 2 (p.T70N) and exon 4 (p.W112R) of the lysozyme gene (LYZ), with both mutations being present on the same allele. This type of hereditary ALys amyloidosis is characterized by extended amyloid deposits in the upper gastrointestinal tract, entire colon, and kidney, leading to gastrointestinal bleeding. Both mutations are probably effective in disease manifestation. The novel mutation at position 112 in the mature protein is located within the alpha-helical domain of the protein and therefore outside the cluster of residues that has so far been implicated in ALys amyloidosis. Taken together with the p.T70N mutation, this results in a lysozyme species where the correct folding of various protein domains is probably impaired and increases the propensity of amyloid fibril formation. Interestingly, this form of ALys amyloidosis is also characterized by the occurrence of proteolytic fragments of lysozyme in the amyloid deposits.     

Isolation and Characterization of a x Amyloid Fibril Protein

The fibril in primary amyloidosis (AL) is composed of a monoclonal light chain or portions thereof. No unique primary structure has been identified that predisposes certain light chains to form amyloid fibrils. Currently, classification of amyloidosis is based on the biochemistry of the amyloid fibril. We determined the NH2-terminal sequence of an amyloid fibril and found it to be of the kappa I immunoglobulin subgroup. No structural alterations were detected to account for the conversion of the light-chain fragment to an amyloid fibril. Antiserum produced to the fibril protein did not react in immunodiffusion with purified LEP or MAG antigens, which are kappa I proteins. This antiserum may be directed to antigenic sites unique to the immunizing protein and is unable to recognize homologous proteins, rendering it unsuitable for immunochemical identification of amyloid deposits of light-chain origin. PAG represents the 10th reported variable kappa I amyloid fibril protein subjected to partial sequence analysis. Antisera that recognize antigenic determinants present in all members of an immunoglobulin subgroup need further development.     

Hereditary Amyloid Cardiomyopathy Caused by a Variant Apolipoprotein A1

Autosomal dominant hereditary amyloidosis with a unique cutaneous and cardiac presentation and death from heart failure by the sixth or seventh decade was found to be associated with a previously unreported point mutation (thymine to cytosine, nt 1389) in exon 4 of the apolipoprotein A1 (apoA1) gene. The predicted substitution of proline for leucine at amino acid position 90 was confirmed by structural analysis of amyloid protein isolated from cardiac deposits of amyloid. The subunit protein is composed exclusively of NH₂-terminal fragments of the variant apoA1 with the longest ending at residue 94 in the wild-type sequence. Amyloid fibrils derived from four previously described apoA1 variants are composed of similar fragments with carboxyl-terminal heterogeneity, but contrary to those variants, which all carry one extra positive charge, the substitution Leu90Pro does not result in any charge modification. It is unlikely, therefore, that amyloid fibril formation is related to change of charge for a specific residue of the precursor protein. This is in agreement with studies on transthyretin amyloidosis in which no unifying factor such as change of charge for amino acid residues has been noted.     

Primary amyloidosis A. Immunohistochemical and biochemical characterization.

Primary "idiopathic" amyloidosis is usually related to immunoglobulin light chain (AL) associated with immunocytic dyscrasias, while secondary "reactive" amyloidosis (AA) is related to serum amyloid A protein (SAA) and typically occurs with chronic inflammation, malignancy, or familial Mediterranean fever. In the present study, amyloid fibril protein extracted from frozen and paraffin-embedded tissue from a patient (CAR) with primary systemic amyloidosis proved to be AA protein by immunohistochemical, immunochemical, and amino terminal sequence. Extracts from both frozen and formalin-fixed paraffin-embedded kidney and spleen yielded similar monomers and dimers of the AA protein. The additional high-molecular-weight bands and a distinct 12,000-dalton fragment in the amyloid protein extracted from the formalin-fixed paraffin-embedded lung suggest that different processing of proteins, ie, by polymerization and/or degradation, may occur in different organs.     

Hepatic amyloidosis. A histopathologic analysis of primary (AL) and secondary (AA) forms.

The liver is a major site of amyloid deposition. The spectrum of histopathologic changes in the liver was studied in 38 patients with systemic amyloidosis (25 with primary or myeloma-associated amyloidosis [AL] and 13 with secondary, reactive [AA] amyloidosis). Overall architectural distortion, alterations of portal triads, as well as predilection for topographic deposition in the parenchyma and/or blood vessel walls were noted. Significant histopathologic differences in AL or AA amyloid liver involvement included 1) portal fibrosis, seen in 7 of 25 (28%) AL patients and 8 of 13 (62%) AA patients (P = 0.05), 2) parenchymal amyloid deposition in 25 of 25 (100%) AL amyloid and 10 of 13 (77%) AA amyloid patients (P = 0.04), and 3) vascular amyloid deposition found in 17 of 25 (68%) with AL amyloid and 13 of 13 (100%) patients with AA amyloid (P = 0.02). These data vary from the widely held concept that deposition of amyloid is predominantly vascular in the AL form and parenchymal in amyloid AA. Clearly, however, in individual cases significant overlap occurred, and characterization of amyloid types based on morphologic distribution of amyloid deposits may be possible in only a minority of cases. In most cases, differentiation of amyloid AL and amyloid AA forms requires clinical, histochemical, immunochemical, and sometimes more elaborate laboratory amino acid sequence studies for accurate identification.